To 50 μL of plasma, 5 μL of CD14 PE and 5 μL of CD16 PECy7 (Biolegend, San Diego, CA) were added. Simultaneously, compensation beads (eBioscience, San Diego, CA) were incubated with the antibodies. Isotype controls for the antibodies of interest (IgG1 k, PE and IgG1 k PECy7) were included. Samples were analyzed by flow cytometry (BD Biosciences, LSRII, San Jose, CA). The instrument was cleaned before analyzing each set of samples. Filtered water, phosphate-buffered saline, and antibodies alone were also tested to confirm elimination of background. Size beads (sub-micron particle size reference kit, ThermoFisher Scientific, .1, .2, .5 and 1 μm) functioned as size markers, and a log scale for forward scatter and side scatter parameters was used. Flow cytometry gates were set at <1 μm and .2 μm. Flow analysis was performed with FlowJo software (FlowJo, LLC, Ashland, OR).
Cd14 pe
Manufactured by BioLegend
Sourced in United States
CD14-PE is a fluorochrome-conjugated monoclonal antibody that recognizes the CD14 antigen. CD14 is a glycosylphosphatidylinositol-anchored protein expressed on the surface of monocytes, macrophages, and granulocytes. It serves as a receptor for lipopolysaccharide (LPS) and plays a role in the innate immune response to bacterial infections.
Automatically generated - may contain errors
Lab products found in correlation
Cd34 pe, by BioLegend (4 mentions)
Cd16 apc, by BioLegend (3 mentions)
Cd11c apc, by BioLegend (3 mentions)
Cd19 pe, by BioLegend (3 mentions)
Cd45 pe, by BioLegend (3 mentions)
Apc anti human cd16 antibody, by BioLegend (2 mentions)
Igg2a pe, by Thermo Fisher Scientific (2 mentions)
Igg2a pe, by BioLegend (2 mentions)
Igg1 apc, by BioLegend (2 mentions)
Anti human cd11b apc antibody, by BioLegend (2 mentions)
F4 80 pe, by Thermo Fisher Scientific (2 mentions)
Facscanto 2 cell analyzer, by BD (2 mentions)
Cd45 fitc, by BioLegend (2 mentions)
Cd25 apc, by BioLegend (2 mentions)
Brefeldin a, by BioLegend (2 mentions)
Cd16 apc cy7, by BioLegend (2 mentions)
Ccr2 percp cy5, by BioLegend (2 mentions)
Cd8 apc, by BioLegend (2 mentions)
Facsdiva, by BD (2 mentions)
Facsaria, by BD (2 mentions)
26 protocols using cd14 pe
1
Plasma Immunophenotyping by Flow Cytometry
2
Flow Cytometry Analysis of Macrophages
3
Quantifying Monocytes and Viability in SVF
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Following SVF isolation from the whole WAT, the pellet enriched for the SVF is resuspended and used either for monocyte/macrophage detection and quantification or for assessing cell viability in response to treatment with metformin. Monocyte/macrophage detection and quantification was assessed by staining with commercially validated antibodies CD14-PE (BioLegend; Cat#301806, clone:M5E2), CD45-FITC (BioLegend; Cat#304005, clone: HI30), CD206-BV-421 (BioLegend; Cat#321125, clone: 15-2), CD3-APC (BioLegend; Cat#317318, clone: OKT3). All antibodies were diluted 1:100 in 100 μl FACS buffer (1% BSA, 5 mM EDTA, 0.05% Sodium Azide in PBS) and incubated at 4 °C, for 30 min. SVF was then centrifuged at 300 g for 5 min and resuspended in Dead/Live stain diluted 1:200 (Thermo Fisher Scientfic; Cat#L34975) and incubated for further 10 min on ice. Cells were then centrifuged at 300 g for 5 min and fixed in 1% PFA before flow cytometry. For measuring cell viability, SVF was stained with APC- Annexin V (1:30 dilution) and propidium iodide (PI; 1:50 dilution) according to the manufacturer's instructions (BioLegend; Cat#79998). All samples were acquired on a LSRFortessa flow cytometer (BD Biosciences, USA) and data were analysed with FlowJo software, version 10 (Tree Star Inc. Ashland, OR, USA).
4
Flow Cytometry Analysis of Macrophages
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The flow cytometry was carried out using the BD FACSCanto™ II Cell Analyzer (BD Bioscience). The analysis of THP-1 macrophages, human classical monocytes, and Kupffer cells was performed as described previously with some modifications (Tian et al., 2016 (link)). Briefly, cells were collected and incubated with primary antibodies IgG2a-PE, IgG1-APC, CD14-PE, APC anti-human CD11b antibody (Biolegend, #301310), APC anti-human CD16 antibody (Biolegend, #360705), F4/80-PE (eBioscience, #12-4801-82), or IgG2a-PE (eBioscience, #12-4321-80) at 4°C for 30 minutes. Cells were rinsed with PBS three times before the flow cytometry analysis. Data were analyzed by FlowJo 10 software (BD Bioscience)
5
Flow Cytometry Analysis of Lung Tissue Immune Cells
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To validate results from the GSVA immune enrichment in the LTRC, we used flow cytometry, a non-mRNA related method. Fresh lung tissue samples of IPF patients undergoing bilateral lung transplant at the University of Pittsburgh (USA) were washed with PBS and enzymatically digested as previously described [34 (link)]. Lung homogenates included multiple areas of the same lung lobe, ensuring the representability of the sample to address patient’s heterogeneity. Lung tissue homogenates (106 cells) were then stained 5 min with the viability staining (Fixable viability-Alexa600, BD, USA) and 30 min at 4ºC in the dark with the following conjugated monoclonal antibodies CD3-PECy5.5, CD45-Alexa700, CD16-BV412, CD56-FITC, CD8-V500, CD4-APC-Cy7, CD19-BV650 (BD, USA) and CD14-PE (BioLegend, USA). A minimum of 5 × 105 cells per sample were acquired in a FACS LSRII (BD Biosciences, USA), and data was analyzed using FlowJo v10 (FlowJo LLC, USA). Immune cell populations were determined using the gating strategy depicted in Additional file 1 : Fig. S1.
6
Immunophenotyping of GMSCs and Splenocytes
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GMSCs were collected and suspended in cell staining buffer (0.5% BSA in PBS with 2 mM EDTA) followed by incubation with CD14 (PE), CD19 (PerCP-Cy5.5), CD29 (APC), CD34 (PE), CD44 (FITC), CD45 (PE), CD73 (PE), CD90 (FITC), and CD105 (PE) antibodies (Biolegend) in the dark at room temperature for 30 min. For intracellular staining, splenocytes from mice were first stained with CD3 (APC/Cy7), CD4 (FITC), and CD25 (APC) antibodies (Biolegend) and then fixed, permeabilized, and stained intracellularly for IL-4 (PE/Cy7) and IL-17 (PE). After staining, cells were washed twice with PBS and submitted to flow cytometric analysis (BD). Data were analyzed using the FlowJo 7.6 software.
7
Isolation and Culture of Mouse Mammary Epithelial Cells
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Primary mouse MEC cultures were isolated from both inguinal and/or thoracic mammary glands and prepared as described [17 (link)]. MEC suspensions and flow cytometry were performed as previously described [40 (link)]. Antibodies against mouse antigens were purchased from Biolegend and included CD24-PB (#101820), CD31 (#102410), CD45 (#103112), and Ter119 (#116212) conjugated to APC, CD29–FITC (#102206), and CD14-PE (#123309). Cells were sorted on a FACSAria or FACSDiva (BD PharMingen) and manually counted prior to plating on irradiated fibroblast feeders as described [40 (link)]. After 7 days, colonies were fixed and stained with Giemsa and manually counted. To induce R26creERT2-mediated deletion, 0.1 μM 4OHT was added to culture medium for 20 hours on day 1 of culture.
8
Immune Cell Isolation from Surgical Specimens
9
Evaluating Immune Responses to Extracellular Vesicles
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Healthy EVs with or without LL37 were used to stimulate healthy PBMCs for determining IP10, IL1β and IFNα positive cells. Healthy PBMCs were stimulated with EV or EV/LL37 complex for 6 h and 16 h in the presence of Brefeldin-A (5 µg ml–1, Biolegend, Cat#420601) for IL1β and IP10 analysis, respectively. Of note, Brefeldin-A was added at t = 0 and t = 6th hour of incubation for IL1β and for IP10, respectively. The cells were then fixed with Fix & Perm Medium A (Invitrogen, Cat# GAS001S100). Cells were first stained with CD14-PE (Biolegend, Cat#325606) and then to detect the level of intracellular IL1β or IP10 were stained wither with IL1β-FITC (e-Biosciences, Cat# 12-7018-81) or IP10-Biotin (BD, Cat# 555048) then SA-FITC (Biolegend, Cat# 405202) in the presence of saponin (0.1%).
IFNα secreting cells were analysed with Miltenyi Biotech’s (San Diego, CA, USA) IFNα Secretion Assay and Detection Kit (PE-labelled, Miltenyi, Cat# 130-094-161) from PBMCs in accordance with kit instructions. Additionally, in order to identify IFNα secreting pDCs, cells were stained with BDCA-2-FITC (Biolegend, Cat# 354208) and then analysed by BD Accuri C6 flow cytometer.
IFNα secreting cells were analysed with Miltenyi Biotech’s (San Diego, CA, USA) IFNα Secretion Assay and Detection Kit (PE-labelled, Miltenyi, Cat# 130-094-161) from PBMCs in accordance with kit instructions. Additionally, in order to identify IFNα secreting pDCs, cells were stained with BDCA-2-FITC (Biolegend, Cat# 354208) and then analysed by BD Accuri C6 flow cytometer.
10
Profiling Monocyte Subsets by Flow Cytometry
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PBMCs were incubated with mixtures of fluorochrome-conjugated antibodies, and identified by flow cytometry. Data acquisition and analysis were performed using a BD CANTO II Flow Cytometer and BD FACS DIVA software (BD Biosciences, Flanklin Lakes, New Jersey). Dead cells were distinguished by 7-amino-actinomycin D (7-AAD, BD Biosciences) staining. Cells were not permeabilized for intra-staining. To identify monocyte lineage cells, the surface markers CD14-PE (Biolegend, San Diego, California, clone HCD14) and CD16-APC/Cy7 (Biolegend, clone 3G8) were used. Pro-inflammatory and anti-inflammatory phenotypes were characterized using S100A9-FITC (BioRad, Hercules, California, clone MAC387) and CD163-APC (Biolegend, clone RM3/1) antibodies (Supplementary Table). S100A9 and CD163 expression levels were determined using a positive dataset for each antibody, identified using a matched concentration of mouse IgG1 kappa isotype control for fluorochrome color (Biolegend, clone MOPC21).
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